Parameter Optimization for Interaction between C-Terminal Domains of HIV-1 Capsid Protein

HIV-1 capsid proteins (CAs) assemble into a capsid that encloses the viral RNA. The binding between a pair of C-terminal domains (CTDs) constitutes a major interface in both the CA dimers and the large CA assemblies. Here, we attempt to use a general residue-level coarse-grained model to describe the interaction between two isolated CTDs in Monte Carlo simulations. With the standard parameters that depend only on the residue types, the model predicts a much weaker binding in comparison to the experiments. Detailed analysis reveals that some Lennard-Jones parameters are not compatible with the experimental CTD dimer structure, thus resulting in an unfavorable interaction energy. To improve the model for the CTD binding, we introduce ad hoc modifications to a small number of Lennard-Jones parameters for some specific pairs of residues at the binding interface. Through a series of extensive Monte Carlo simulations, we identify the optimal parameters for the CTD–CTD interactions. With the refined model parameters, both the binding affinity (with a dissociation constant of 13 ± 2 μM) and the binding mode are in good agreement with the experimental data. This study demonstrates that the general interaction model based on the Lennard-Jones potential, with some modest adjustment of the parameters for key residues, could correctly reproduce the reversible protein binding, thus potentially applicable for simulating the thermodynamics of the CA assemblies

Monte Carlo Simulations of HIV Capsid Protein Homodimer

Capsid protein (CA) is the building block of virus coats. To help understand how the HIV CA proteins self-organize into large assemblies of various shapes, we aim to computationally evaluate the binding affinity and interfaces in a CA homodimer. We model the N- and C-terminal domains (NTD and CTD) of the CA as rigid bodies and treat the five-residue loop between the two domains as a flexible linker. We adopt a transferrable residue-level coarse-grained energy function to describe the interactions between the protein domains. In seven extensive Monte Carlo simulations with different volumes, a large number of binding/unbinding transitions between the two CA proteins are observed, thus allowing a reliable estimation of the equilibrium probabilities for the dimeric vs monomeric forms. The obtained dissociation constant for the CA homodimer from our simulations, 20–25 μM, is in reasonable agreement with experimental measurement. A wide range of binding interfaces, primarily between the NTDs, are identified in the simulations. Although some observed bound structures here closely resemble the major binding interfaces in the capsid assembly, they are statistically insignificant in our simulation trajectories. Our results suggest that although the general purpose energy functions adopted here could reasonably reproduce the overall binding affinity for the CA homodimer, further adjustment would be needed to accurately represent the relative strength of individual binding interfaces

Highly Mutable Linker Regions Regulate HIV-1 Rev Function and Stability.

HIV-1 Rev is an essential viral regulatory protein that facilitates the nuclear export of intron-containing viral mRNAs. It is organized into structured, functionally well-characterized motifs joined by less understood linker regions. Our recent competitive deep mutational scanning study confirmed many known constraints in Rev's established motifs, but also identified positions of mutational plasticity, most notably in surrounding linker regions. Here, we probe the mutational limits of these linkers by testing the activities of multiple truncation and mass substitution mutations. We find that these regions possess previously unknown structural, functional or regulatory roles, not apparent from systematic point mutational approaches. Specifically, the N- and C-termini of Rev contribute to protein stability; mutations in a turn that connects the two main helices of Rev have different effects in different contexts; and a linker region which connects the second helix of Rev to its nuclear export sequence has structural requirements for function. Thus, Rev function extends beyond its characterized motifs, and is tuned by determinants within seemingly plastic portions of its sequence. Additionally, Rev's ability to tolerate many of these massive truncations and substitutions illustrates the overall mutational and functional robustness inherent in this viral protein

Hybrid Approaches to Structural Characterization of Conformational Ensembles of Complex Macromolecular Systems Combining NMR Residual Dipolar Couplings and Solution X‑ray Scattering

Solving structures or structural ensembles of large macromolecular systems in solution poses a challenging problem. While NMR provides structural information at atomic resolution, increased spectral complexity, chemical shift overlap, and short transverse relaxation times (associated with slow tumbling) render application of the usual techniques that have been so successful for medium sized systems (\u3c50 \u3ekDa) difficult. Solution X-ray scattering, on the other hand, is not limited by molecular weight but only provides low resolution structural information related to the overall shape and size of the system under investigation. Here we review how combining atomic resolution structures of smaller domains with sparse experimental data afforded by NMR residual dipolar couplings (which yield both orientational and shape information) and solution X-ray scattering data in rigid-body simulated annealing calculations provides a powerful approach for investigating the structural aspects of conformational dynamics in large multidomain proteins. The application of this hybrid methodology is illustrated for the 128 kDa dimer of bacterial Enzyme I which exists in a variety of open and closed states that are sampled at various points in the catalytic cycles, and for the capsid protein of the human immunodeficiency virus

Mathematical models for HIV-1 viral capsid structure and assembly

Includes bibliographical references.2015 Summer.HIV-1 (human immunodeficiency virus type 1) is a retrovirus that causes the acquired immunodeficiency syndrome (AIDS). This infectious disease has high mortality rates, encouraging HIV-1 to receive extensive research interest from scientists of multiple disciplines. Group-specific antigen (Gag) polyprotein precursor is the major structural component of HIV. This protein has 4 major domains, one of which is called the capsid (CA). These proteins join together to create the peculiar structure of HIV-1 virions. It is known that retrovirus capsid arrangements represent a fullerene-like structure. These caged polyhedral arrangements are built entirely from hexamers (6 joined proteins) and exactly 12 pentamers (5 proteins) by the Euler theorem. Different distributions of these 12 pentamers result in icosahedral, tubular, or the unique HIV-1 conical shaped capsids. In order to gain insight into the distinctive structure of the HIV capsid, we develop and analyze mathematical models to help understand the underlying biological mechanisms in the formation of viral capsids. The pentamer clusters introduce declination and hence curvature on the capsids. The HIV-1 capsid structure follows a (5,7)-cone pattern, with 5 pentamers in the narrow end and 7 in the broad end. We show that the curvature concentration at the narrow end is about five times higher than that at the broad end. This leads to a conclusion that the narrow end is the weakest part on the HIV-1 capsid and a conjecture that “the narrow end closes last during maturation but opens first during entry into a host cell.” Models for icosahedral capsids are established and well-received, but models for tubular and conical capsids need further investigation. We propose new models for the tubular and conical capsid based on an extension of the Caspar-Klug quasi-equivalence theory. In particular, two and three generating vectors are used to characterize respectively the lattice structures of tubular and conical capsids. Comparison with published HIV-1 data demonstrates a good agreement of our modeling results with experimental data. It is known that there are two stages in the viral capsid assembly: nucleation (formation of a nuclei: hexamers) and elongation (building the closed shell). We develop a kinetic model for modeling HIV-1 viral capsid nucleation using a 6-species dynamical system. Numerical simulations of capsid protein (CA) multimer concentrations closely match experimental data. Sensitivity and elasticity analysis of CA multimer concentrations with respect to the association and disassociation rates further reveals the importance of CA dimers in the nucleation stage of viral capsid self-assembly

Structure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer

Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Here, visualized by cryo-electron tomography and subtomogram averaging, mature capsid-like particles show an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 have opposing effects on CA in vitro assembly, inducing formation of T = 1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles reveal that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles

Improvement of viral fusion inhibitor enfuvirtide efficacy by conjugation with membrane anchoring lipids

The human immunodeficiency virus type 1 (HIV-1) is a highly pathogenic and evasive virus, for which no cure has yet been achieved. The majority of the antiretroviral drugs developed over the years against this infection target key enzymes in HIV life cycle, such as reverse transcriptase, integrase and protease. Fusion of viral and host cell membranes is a crucial step in virus infectivity;therefore,the development of viral entry inhibitors has great advantages over conventional drugs, since they prevent the release of the viral content into the host cell. Previous studies showed that the antiviral activity of HIV-1inhibitor peptides is increased bythe addition of cholesterol and polyethylene glycol (PEG). The aim of the present work isto characterizethe interaction of enfuvirtide derived molecules by conjugation with cholesterol,palmitic acid or α-tocopherol as lipid anchors and PEG as spacer, with biomembrane model systems and human blood cells.Fluorescence spectroscopymethodologies, including membrane partition and fluorescence quenching assays,demonstrated that conjugation with lipids increasesthe peptides ability to interact with membranes of different compositions. In addition, the depth of peptide insertion into the membrane was assessed using lipophilic probes, revealing that the conjugated peptides are located in a more shallow position than the unconjugated one. Moreover, dipole potential assays showed that conjugated peptides exhibit a higher affinity towards cholesterol-rich membranes, as well as human blood cells, than the unconjugated peptide.Altogether, the obtained results indicate that a proper balance between membrane affinity and peptide exposure is required in order to enhance antiviral activity. Therefore, the addition of lipid moieties to an established fusion inhibitor such as enfuvirtide can be a promising strategy against HIV-1

A simple fluorescence based assay for quantification of human immunodeficiency virus particle release

<p>Abstract</p> <p>Background</p> <p>The assembly and release of human immunodeficiency virus (HIV) particles from infected cells represent attractive, but not yet exploited targets for antiretroviral therapy. The availability of simple methods to measure the efficiency of these replication steps in tissue culture would facilitate the identification of host factors essential for these processes as well as the screening for lead compounds acting as specific inhibitors of particle formation. We describe here the development of a rapid cell based assay for quantification of human immunodeficiency virus type 1 (HIV-1) particle assembly and/or release.</p> <p>Results</p> <p>Using a fluorescently labelled HIV-derivative, which carries an eYFP domain within the main viral structural protein Gag in the complete viral protein context, the release of virus like particles could be monitored by directly measuring the fluorescence intensity of the tissue culture supernatant. Intracellular Gag was quantitated in parallel by direct fluorescence analysis of cell lysates, allowing us to normalize for Gag expression efficiency. The assay was validated by comparison with p24 capsid ELISA measurements, a standard method for quantifying HIV-1 particles. Optimization of conditions allowed the robust detection of particle amounts corresponding to 50 ng p24/ml in medium by fluorescence spectroscopy. Further adaptation to a multi-well format rendered the assay suitable for medium or high throughput screening of siRNA libraries to identify host cell factors involved in late stages of HIV replication, as well as for random screening approaches to search for potential inhibitors of HIV-1 assembly or release.</p> <p>Conclusions</p> <p>The fast and simple fluorescence based quantification of HIV particle release yielded reproducible results which were comparable to the well established ELISA measurements, while in addition allowing the parallel determination of intracellular Gag expression. The protocols described here can be used for screening of siRNA libraries or chemical compounds, respectively, for inhibition of HIV in a 96-well format.</p

Development of a Novel Bistable DNA Sensor for Anti-HIV Drug Discovery and Re-engineering of Recombinant Cyclin T1-Tat Protein with SUMO Fusion in Escherichia Coli

This dissertation focuses on the interaction of proteins and nucleic acids and their applications. It includes two projects: I: Development of a Novel Bistable DNA Sensor for Anti-HIV Drug Discovery and II. Re-engineering of Recombinant Cyclin T1-Tat Protein with SUMO Fusion in Escherichia Coli.I: Development of a Novel Bistable DNA Sensor for Anti-HIV Drug Discovery Screening drug compounds targeting HIV-1 NCp7 provide attractive candidates for new anti-retroviral therapeutics because of the highly conserved nature of the zinc fingers in NCp71 in selecting and packaging RNA in the HIV-1 life cycle. The unique 3-segment, reversible switch for high throughput screening (HTS) drug targets will be for the HIV-1 nucleocapsid (NC) protein. The Probe is a natural binding element for the NCp7 protein target or, in our case, an aptamer hairpin with loop sequence, TGTGGT, having a nano-molar affinity (Kd=16nM).2 Toggle is a damaged probe where the target-binding sequences are replaced with other bases. Cover is a mostly complementary strand for the probe and the toggle. A hairpin loop forms around the 5Me-dC-brancher in both the ON and OFF forms. Using Visual OMPTM simulation, the following two potential switch molecules (NM-1 and NM-2) were designed and successfully synthesized using a one-step ligation method with at least 90% purity as judged by mass spectrometry. Then fluorescence measurements using NM-1 and NM-2 with NCp7 protein were analyzed to demonstrate proof-of-principle for 3-segment nucleic acid switches. Increasing [NC] causes a dramatic decrease in CY3 fluorescence. The ON/OFF contrast ratio of NM-1 and NM-2 are 2.6 and 3.2 showing the feasibility of 3-segment switches being used to further modify the design of HTS switches for the HIV-1 NC. II. Re-engineering of Recombinant Cyclin T1-Tat Protein with SUMO Fusion in Escherichia Coli. Due to increasing drug resistance for current antiretroviral therapeutic (ART) treatments, it is important to add drugs targeting other aspects of HIV-1 infection to manage AIDS. Tat (trans-activator of transcription) protein up-regulates the transcription of viral-specific proteins by a factor of 1,0003, which makes Tat a very attractive drug target. High-level expression and purification of recombinant GST-CycT1(249-280)-linker(25aa)-Tat(1-101) fusion protein in Escherichia coli (E. coli) was challenging because CycT1-Tat forms inclusion bodies making it difficult to purify and obtain a high concentration of active CycT1-Tat. A commercially available pET-SUMO cloning vector was introduced for the high-level expression of four CycT1-Tat chimeras, F1-F4. The gene associated with the protein sequence was purchased from IDT and SUMO-CycT1-Tat (F1) fusion plasmid was prepared and transformed in E. coli BL21 (DE3). By inducing with IPTG, His6-SUMO-CycT1-Tat fusion protein was able to promote soluble expression making it more efficient to purify CycT1-Tat (F1) compared to GST fusion tag protein. The molecular weight of purified CycT1-Tat was confirmed with mass spectrometry (MALDI-TOF). For initial characterization of the TAR-CycT1-Tat complex, an Electrophoretic Mobility Shift Assay (EMSA) was carried out with partially purified CycT1-Tat protein using TAR-31 and truncated versions that altered the hairpin loop (TAR-H24) and deleted the bulge loop (TAR-B25). A TAR-CycT1-Tat complex was formed and a band shift was observed in the binding of CycT1-Tat to TAR-31 and a lower affinity complex with TAR-B25, but TAR-H24 did not bind